Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control
The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exo...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/13/9/1589 |
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| author | Teruaki Tozaki Aoi Ohnuma Kotono Nakamura Kazuki Hano Masaki Takasu Yuji Takahashi Norihisa Tamura Fumio Sato Kyo Shimizu Mio Kikuchi Taichiro Ishige Hironaga Kakoi Kei-ichi Hirota Natasha A. Hamilton Shun-ichi Nagata |
| author_facet | Teruaki Tozaki Aoi Ohnuma Kotono Nakamura Kazuki Hano Masaki Takasu Yuji Takahashi Norihisa Tamura Fumio Sato Kyo Shimizu Mio Kikuchi Taichiro Ishige Hironaga Kakoi Kei-ichi Hirota Natasha A. Hamilton Shun-ichi Nagata |
| author_sort | Teruaki Tozaki |
| collection | DOAJ |
| description | The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the <i>myostatin</i> gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses. |
| first_indexed | 2024-03-09T23:56:28Z |
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| id | doaj.art-747788df561241559e70ee22d958afe4 |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-09T23:56:28Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-747788df561241559e70ee22d958afe42023-11-23T16:24:40ZengMDPI AGGenes2073-44252022-09-01139158910.3390/genes13091589Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping ControlTeruaki Tozaki0Aoi Ohnuma1Kotono Nakamura2Kazuki Hano3Masaki Takasu4Yuji Takahashi5Norihisa Tamura6Fumio Sato7Kyo Shimizu8Mio Kikuchi9Taichiro Ishige10Hironaga Kakoi11Kei-ichi Hirota12Natasha A. Hamilton13Shun-ichi Nagata14Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, JapanGenetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, JapanDepartment of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu 501-1193, JapanDepartment of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu 501-1193, JapanDepartment of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu 501-1193, JapanEquine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke 329-0412, JapanEquine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke 329-0412, JapanEquine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke 329-0412, JapanRegistration Department, Japan Association for International Racing and Stud Book, 4-5-4, Shimbashi, Minato, Tokyo 105-0004, JapanGenetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, JapanGenetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, JapanGenetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, JapanGenetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, JapanEquine Genetics Research Centre, Racing Australia, 2 Randwick Way, Scone, NSW 2337, AustraliaGenetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, JapanThe creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the <i>myostatin</i> gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.https://www.mdpi.com/2073-4425/13/9/1589amplicon sequencinggene dopinggene editinghorsethoroughbred |
| spellingShingle | Teruaki Tozaki Aoi Ohnuma Kotono Nakamura Kazuki Hano Masaki Takasu Yuji Takahashi Norihisa Tamura Fumio Sato Kyo Shimizu Mio Kikuchi Taichiro Ishige Hironaga Kakoi Kei-ichi Hirota Natasha A. Hamilton Shun-ichi Nagata Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control Genes amplicon sequencing gene doping gene editing horse thoroughbred |
| title | Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control |
| title_full | Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control |
| title_fullStr | Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control |
| title_full_unstemmed | Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control |
| title_short | Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control |
| title_sort | detection of indiscriminate genetic manipulation in thoroughbred racehorses by targeted resequencing for gene doping control |
| topic | amplicon sequencing gene doping gene editing horse thoroughbred |
| url | https://www.mdpi.com/2073-4425/13/9/1589 |
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